Study On The Relationship Between SLP-2 Expression And The Cells Proliferation And Invasion Of Colon Cancer And Its Molecular Mechanism

Colon cancer(CC)is a common malignant tumor in the digestive tract.Because it has more common pathophysiological features with rectal cancer,they are commonly referred to as colorectal cancer(CRC).In recent years,the incidence and mortality of CC have showed a significant upward trend,and there is a trend of getting younger.CC has already had a serious impact on the life and health of our residents.With the development of colonoscopy,the early diagnosis and treatment of CC has made some progress,but,the survival rate of patients with advanced CC has not been substantially improved.Therefore,it is of great practical significance to study the pathogenesis and transfer mechanism of CC.SLP-2(stomatin-like protein 2)is a member of the stomatin gene superfamily,and is first discovered by the pathology department of Yale university in 2000.Studies have shown that SLP-2protein is a membrane protein,which is an important part of the cytoskeleton,and may be involved in the regulation of cell membrane ion channels.In addition,SLP-2 protein is also a mitochondrial related protein,which exists in the mitochondria of cell.It can regulate the stability of mitochondrial membrane associated proteins and affect the synthesis of ATP.In recent years,numbers of studies have shown that SLP-2 is associated with the development of malignant tumors.For example,esophageal squamous cell carcinoma,breast cancer,cervical cancer,and so on.Little is known about the effect of SLP-2 on CC,and it is not clear that SLP-2 play a cancer suppression or promotion role in the development of CC,so it is necessary to carry out further study.We studied the function of SLP-2 in CC by the following 5 parts: First,the expression levels of SLP-2mRNA and protein in CC and adjacent normal tissues were detected by real-time quantitative PCR(qRT-PCR)and Western blot to investigate the relationship between SLP-2 expression and differentiation of CC.Second,we examined the SLP-2 mRNA and protein expression levels of six common CC cells by qRT-PCR and Western blot to investigate the relationship between SLP-2 expression and the differentiation degree of colon cancer cells in the laboratory.Third,knocking down the expression level of SLP-2 in colon cancer cells by siRNA interference assay,then we performed MTT assay,flow cytometry,Western blot and qRT-PCR to test the influence of SLP-2 on CC cell proliferation and cell cycle.Fourth,knocking down the expression level of SLP-2 in colon cancer cells by siRNA interference assay,then we performed cell scratch test,transwell migration assay,Western blot and qRT-PCR to test the influence of SLP-2 on CC cell migration and invasion.Finally,Western blot and qRT-PCR were used to detect the effect of SLP-2 on ?-catenin and its downstream signal pathway,and the molecular mechanism affecting the proliferation,migration and invasion of colon cancer cells was discussed.Part 1 Relationship between the expression of SLP-2 in colonicalcancerous tissue and colon cancerObjective: To analyze the SLP-2 expression in colon cancer and adjacent normal tissues;To explore the relationship between SLP-2 expression and differentiation level of colon cancer tissues.Methods: 30 colon cancer patients who underwent surgical removal of the primary tumors between January 2017 and June 2018 at the Fourth Hospital of Hebei Medical University were recruited.From each of the CC patient,tumor tissues and matched adjacent non-cancerous tissue samples(more than 2 cm from the tumor edge with no carcinoma tissue and atypical hyperplasia detected under microscope)were collected for SLP-2 expression.Then,the expression of SLP-2 mRNA and protein of the colon cancer tissues and adjacent normal tissues were detected by qRT-PCR and Western blot assay.Results:1.Expression of SLP-2 mRNA in colon cancer tissues and adjacent tissuesCompared with the matched adjacent normal tissues,the results of qRTPCR showed that the SLP-2 mRNA expression level in tumor tissues was significantly higher(P < 0.01).The expression level of low differentiated tissue was higher than that of high differentiated tissue(P < 0.01).2.Expression of SLP-2 protein in colon cancer tissues and adjacent tissuesCompared with the matched adjacent normal tissues,the results of Western blot showed that the SLP-2 protein expression level in tumor tissues was significantly higher(P < 0.01).The expression level of low differentiated tissue was higher than that of high differentiated tissue(P < 0.01).Conclusions: The expression level of SLP-2 in colon cancer tissues is significantly higher than that in adjacent normal tissues,and its expression is related to the degree of tissue differentiation of colon cancer,suggesting that it plays an important role in the occurrence and evolution of colon cancer and has clinical value as a potential target for early diagnosis and treatment of colon cancer patients.Part 2 Expression differences of SLP-2 in colon cancer cell linesObjective: To investigate the relationship between SLP-2 mRNA and protein and the degree of differentiation of colon cancer cells in six colon cancer cell lines.Methods: Six common colon cancer cell lines(HT29,SW480,SW620,LOVO,caco-2,HCT116)and normal colonic epithelial cells(NCM460)were selected.The expression levels of SLP-2 mRNA and protein were detected by qRT-PCR and Western blot to determine the relationship between SLP-2 mRNA and protein and the differentiation degree of colon cancer cells.Results:1.Expression of SLP-2 mRNA in colon cancer cell lines with different differentiation levelsThe results of qRT-PCR showed that the expression level of SLP-2 mRNA was negatively correlated with the degree of differentiation of colon cancer cells,that is,with the decrease of the degree of differentiation,the expression level of SLP-2 mRNA was gradually increased,and SLP2 mRNA was most highly expressed in SW620 cells,with statistically significant differences(P < 0.01).2.Expression of SLP-2 protein in colon cancer cell lines with different differentiation levelsThe results of Western blot showed that the expression level of SLP-2 protein was negatively correlated with the degree of differentiation of colon cancer cells,that is,with the decrease of the degree of differentiation,the expression level of SLP-2 protein was gradually increased,and SLP-2 protein was most highly expressed in SW620 cells,with statistically significant differences(P < 0.01).Conclusions: The mRNA and protein expression of SLP-2 were different in different colon cancer cell lines,suggesting that the expression level of SLP-2 may be closely related to the biological functions of colon cancer cells,such as differentiation,proliferation,migration and invasion.Part 3 Effect of silencing SLP-2 expression on proliferation and cellcycle of colon cancer cell line-SW620Objective: To investigate the effects of SLP-2 expression on proliferation and cell cycle of colon cancer cells by siRNA transfection.Methods: Three SLP-2 mRNA-specific siRNA sequences were designed and ordered:(SLP-2-si-RNA-1,5'-GCAGAGUCUCAAGGAAAUUtt-3'),(SLP-2-siRNA-2,5'-CGACAAUGUAACUCUGCAAtt-3'),(SLP-2-siRNA-3,5'-G GCCAAGGCUAAAGCUGAAtt-3')and control siRNA(NS-siRNA,5'-GGU CUCACUCC-CCAUAGAGtt-3').SW620 cell lines were transfected with three SLP-2 siRNA sequences high expression plasmid,then SLP-2 mRNA and protein expression changes were observed in different time points after transfection(24,48,72 and 96 h),to make sure the most effective siRNA sequences and minimum effective time.After the successful and stable transfected cell lines were screened and constructed,the effects of silencing SLP-2 on cell proliferation and cell cycle were detected by MTT,flow cytometry,qRT-PCR and Western blot,respectively.Results:1.Effects of SLP-2-siRNAs on SLP-2 mRNA and protein expression in SW620 cellsThe results of qRT-PCR and Western blot showed that,48 h after the transfection,the SLP-2 mRNA and protein were decreased in different degrees with 50 nM different SLP-2-siRNAs sequences into SW620 colon cancer cells.SLP-2-siRNA-2 had the best knockdown effect on SLP-2.However,SLP-2 mRNA and protein expression were not significantly changed after cell transfection with control-siRNA.To determine the minimum effective time of SLP-2 knockdown,we examined the effect on expression of SLP-2 of SLP-2-siRNA-2 transfection in SW620 colon cancer cells at different times(24,48,72 and 96 h).The results of qRT-PCR and Western blot showed that the mRNA and protein expression levels of SLP-2 in SW620 cells were significantly decreased after the transfection of SLP-2-siRNA-2 at 50 nM,and the mRNA and protein expression levels of SLP-2 were decreased by more than 80% at 48,72 and 96 h compared with the control group.In the subsequent experiments,SW620 colon cancer cells transfected with 50 nM SLP-2-siRNA-2 for 48 h were used.2.Effects of SLP-2-siRNA transfection on the proliferation activity of SW620 cellsMTT results showed that SLP-2-siRNA transfected SW620 cells for 48 h,the cell proliferation activity was significantly reduced.Meanwhile,the mRNA and protein expression levels of cell proliferation marker PCNA were significantly decreased(P < 0.01).3.Effects of SLP-2-siRNA transfection on the cell cycle of SW620 cellsFlow cytology showed that SLP-2-siRNA transfected SW620 cells for 48 h,cell cycle progression was inhibited,and the proportion of G0/G1 was significantly increased.At the same time,the mRNA and protein expression levels of cyclin D1 were significantly decreased(P < 0.01).Conclusions: SLP-2-siRNA could effectively reduce the expression of SLP-2 in colon cancer SW620 cell line.Knockdown of SLP-2 expression significantly inhibited the proliferation and cell cycle progression of SW620 in colon cancer cells.Part 4 Effect of silencing SLP-2 expression on invasion and migration ofcolon cancer cellsObjective: To investigate the effects of SLP-2 expression on invasion and migration of colon cancer cells by siRNA transfection.Methods: SLP-2-siRNA-2(5'-CGACAAUGUAACUCUGCAAtt-3')and control siRNA(NS-siRNA,5'-GGUCUCACUCC-CCAUAGAGtt-3')were transfected into SW620 cell lines by SLP-2 siRNA sequences high expression plasmid.After 48 h,Cell scratch assay and Transwell invasion assay were used to detect the effect of silencing SLP-2 on cell invasion and migration ability.qRT-PCR and Western blot were used to detect the effect of silencing SLP-2 on cell invasion and migration related proteins and gene expression(ICAM1,MMP2,MMP7 and MMP9).Results:1.Effect of SLP-2 silencing on migration activity of SW620 cellsCell scratch test results showed that the migration activity of SW620 cells was inhibited significantly after 48 h of SLP-2-siRNA transfection(P<0.05).2.Effect of SLP-2 silencing on the invasive activity of SW620 cellsTranswell invasion assay showed that the invasive activity of SW620 cells was inhibited significantly after 48 h of SLP-2-siRNA transfection(P< 0.05).3.Effects of SLP-2 silencing on invasion and migration related genes and protein expression in SW620 cellsqRT-PCR and western-blot results showed that the mRNA and protein expressions of ICAM1,MMP2,MMP7 and MMP9 in SW620 cells were significantly reduced after transfection with SLP-2-siRNA for 48h(P<0.05),but the expression of TIMP1 and TIMP2 was not significantly affected.Conclusions: Knockdown of SLP-2 significantly inhibited the migration and invasion of colon cancer cell SW620.Part 5 The effect of silencing SLP-2 expression on up-and-downmolecules in the signal pathway of ?-cateninObjective: To investigate the effects of SLP-2 expression on ?-catenin and its downstream signal pathway in colon cancer cells by siRNA transfection and discuss the molecular mechanism of SLP-2 on the proliferation,invasion and migration of colon cancer cells.Methods: SLP-2-siRNA-2(5'-CGACAAUGUAACUCUGCAAtt-3')and control siRNA(NS-siRNA,5'-GGUCUCACUCC-CCAUAGAGtt-3')were transfected into SW620 cell lines by SLP-2 siRNA sequences high expression plasmid.After 48 h,qRT-PCR and Western blot were used to detect the effect of silencing SLP-2 on ?-catenin and its downstream signaling pathway related proteins and gene expression(GSK3?,p-GSK3?,Survivin,cyclin D1 and c-Myc).Results:1.Effects of SLP-2 silencing on the mRNA expression of ?-catenin and its upstream and downstream signaling pathways in SW620 cellsqRT-PCR results showed that the mRNA expression of GSK3? was significantly increased in SW620 cells 48 h after the transfection,and the difference was statistically significant(P<0.05),while the mRNA expression levels of ?-catenin and its downstream molecules(Survivin,cyclin D1 and c-Myc)were significantly decreased(P<0.05).2.Effects of SLP-2 silencing on the protein expression of ?-catenin and its upstream and downstream signaling pathways in SW620 cells.Western-blot results showed that after 48 h of SLP-2-siRNAs transfection,GSK3? protein expression and its phosphorylation level were significantly increased(P<0.05),while protein expression levels of ?-catenin and its downstream molecules(Survivin,cyclin D1 and c-Myc)were significantly decreased(P<0.05).Conclusions: SLP-2 affects the proliferation,migration and invasion of colon cancer cells by affecting the ?-catenin signal pathway.